ASRT 223-25 February 2017, Bangkok

Solanaceous crops and bacterial wilt interactions: overview &

focus on genetics of eggplant resistance

Jacques DINTINGER

Marie-Christine DAUNAY

2 PhD theses

A. Lebeau (2010), S. Salgon (2017)

Solanaceous crops and bacterial wilt interactions: overview &

focus on genetics of eggplant resistance

1. Bacterial wilt diversity

2. Interactions Bacterial Wilt x Solanaceous crops resistances

3. Genetics of eggplant resistance(s) to bacterial wilt

Genetic diversity of Bacterial wilt: Ralstonia solanacearum

very large host range (including Solanaceous crops), persistence in fields

extensive genetic diversity: Ralstonia solanacearum species complex (RSSC)

Clonal lines identified (rep-PCR, PFGE, AFLP, …)

structured in 4 phylotypes (ITS, egl, hrpB, …): I, II (A & B), III et IV (Fegan and Prior, 2005)

Phylotype IIA

Phylotype IIB

Phylotype III

Phylotype IV

Phylotype I

Subdivided in 10 clades (sequence variants (<1,8%) with ecological traits51 sequevars (highly conserved variants (< 1%)

: 2 clades, 11 sequevars

: 3 clades, 8 sequevars

: 1 clade, 16 sequevars

: 1 clade, 11 sequevars

: 3 clades, 4 sequevars

R. sol.Moko

R. pseudo sol.BDB

R. syzygii

Recently classified as 3 species

Phylotype

Mutation rate

Reprod.mode

Demogr.history

I 137.6Highlyrecombin.

Highlyexpanding

III 68.8Highlyrecombin.

Highlyexpanding

IIA 49.4 Recombin. Expanding

IIB 43.3 ClonalWeaklyexpanding

IV 108.2Highlyrecombin.

No expansion

Genetic diversity of bacterial wilt : a worldwide prevalence

Prevalence

-Strains from all phylotypes can wilt eggplant, tomato and pepper-Phylotype I : highest evolutionary potential, worldwide prevalence, highestvirulence diversity

=> Highest ability to adapt to new hosts/resistant cultivars?

Tomato

T1. CRA 66

T2. Okitsu Sozai n°1T3. NC 72 TR 4-4

T4. IRAT L3

T5. Hawaii 7996

T6. TML 46

T7.CLN 1463

T8. R3034

T9. L 285 S. lycop. escul. var. cerasiforme

T10. L 390 (susceptible control)

eggplant

E1. MM853, Dingras multiple purple

E2. MM643, SM6

E3. MM152, Ceylan SM164

E4. MM1752, EG203, Surya

E5. AG91-01, MM931

E6. AG91-25, MM960

E7. MM195 S. linnaeanum (parent of 1rst mappi.pop)

E8. MM738 (parent of 1rst mapping population)

E9. S56B, Terong Bulat Hijau

E10. Florida Market, MM 136 (suscept. control)

Pepper

P1. Narval, PM 1143

P2. PI 322719, PM 687

P3. Cristal Blanco, PM 1022, Pen 79 (C.baccatum)

P4. CM 334, PM 702

P5. 0209-4, BC3F5 (BC3F5[C. annuum x C.chinense])

P6. CA8, PBC631A

P7. MC4, PBC66

P8. Perennial, PM 659

P9. PBC384

P10. Yolo Wonder (susceptible control)

Nominated by crop breeding experts

Tomato: H. Laterrot (INRA), P. Hanson (AVRDC),

J. Scott (Univ. Florida), M.C. Daunay (INRA)

Eggplant: P. Hanson & J.-F. Wang (AVRDC) ,

MC. Daunay (INRA)

Pepper: P. Gniffke (AVRDC), A. Palloix (INRA)

A core-collection (Core-TEP) of worldwide genetic resources resistant to R. solanacearum

12 virulent strains, phylogenetically differentiated

A core-collection (Core-Rs) of R. solanacearum strains

Isolated from: Solanum lycopersicum, Vaccinium sp.Heliconia caribea

Pathogenic to

Phenotype ClusterPercentage of

wilted plant

Colonization

index

Highly resistant 1 ≤ 13,3% ≤ 16,67%

Moderately

resistant 2 ≤ 33,33%

16,67% ≤ … ≤

50%

Intermediate 3 ≤ 43,33% ≥ 43,33%

Susceptible 4 40% ≤ … ≤ 80% ≥ 46,67%

Highly

susceptible 5 ≥ 80% ≥ 86,67%

phenotype statistical partitioning (based on interactions Core-TEP x Core-Rs)

• partially resistant

• latently infected

Colonisation index (%)

% wilted

plants

veryrésistant

Moderately susceptible

Moderatelyrésistant

Very susceptible

Partiallyresistant

Latent infection

6 phenotypes defined

Latent

infection

very

resistant

Moderately

susceptible

Moderately

resistant

Very

susceptible

Partially

résistant

Interactions BW diversity X solanaceous crops resistance diversityStrains selected for genetic studies

Strain Alternative name Country AG91-25 MM738

CMR134 RUN215, CFBP7058 Cameroon 2 4

PSS366 RUN155 Taiwan 1 4

PSS4 RUN157, CIP410 Taiwan 5 5

GMI1000 RUN54, JS753 French Guyana 3.1 5

CFBP2957 RUN36, MT5 Martinique 2 4

CMR34 RUN147, CFBP7029 Cameroon 5 5

CFBP3059 RUN39, JS904 Burkina Faso 4 5

TO10 RUN969 Thaïland na na

1

2

3.1

4

5

highly resistant

moderately resistant

partially resistant

moderately susceptible

highly susceptible

Dissection of the resistance in eggplant AG91-25(breeding line of complex pedigree, involving S. aethiopicum)

Resistance evaluation: protocol

- 180 RILs population Inoculated through irrigation system- 2 greenhouses = 2 replications- 2-season repeat tests for each strain

- Scoring plants according a scale 0 to 4- Evaluation of plants 4 to 5 weeks

post-inoculation- Latent infection tested at the end of

trials

Variables analyzed :

Disease score (SCO): Rating means for each line

Wilting rate (W): % plants with rating ≥ 1

Colonization index (CI): % colonized plants

AUDPC: Area under disease progress curve

AG91-25 (0,038)

AG91-25 (0,041)

MM738 (0,56)

MM738 (0,45)

AG91-25 (0,35)

AG91-25 (0,40)

MM738 (0,68)

MM738 (0,73)

AG91-25 (0,12)

MM738 (0,16)

MM738 (0,30)

MM738 (0,54)

MM738 (0,28)

AG91-25 (0)AG91-25 (0)AG91-25 (0)

Resistance evaluation: results

Three different results with Waudpc variable:

Continuous distributions of RILs population. High differences of resistance between AG91-25 (R) and MM738 (S) (CFBP3059, CFBP2957,

PSS4, TO10)

Quantitative variable controlled by QTLs

Kernel density plots of the variable Waudpc

Continuous distribution but phenotype of parental lines are very close (CMR34)

Distribution due to environmental effects?

Discontinuous distribution with 2 discrete classes : Resistant /susceptible (GMI1000,

PSS3666, CMR134).

Qualitative variable controlled by major gene(s)

Genotyping by sequencing (GBS)

DNA extraction from eggplant population

51 2

DNA digestion with ApeKIenzyme

3

Multiplexing 96 samples with unique barcode

4

PCR amplification to obtain GBS “library”

Sequencing with illuminatechnology

Generation of SNPs

1 Quality control of raw data

2 Clean and demultiplex by barcode

3

Reference eggplant genome

“Denovo”

Stack 1 Stack 2 Stack 3 Stack X

Pipeline Stacks Stacks from parental lines assembled in loci in a catalog

Stacks from population compared to catalog and SNP calling

4 5

Results: A new densified genetic map

Genetic map with 14 LG corresponding to the 12 chromosomes of eggplant

Total map length of 1518 cM (Reference genetic map = 1281 cM) with 1036 markers (867 SNPs)

LG1= E01_ALG2= E02 LG3= E03LG4= E04 LG5= E05_A LG6= E06 LG7= E07 LG8= E08 LG9= E09LG10= E10 LG11= E11 LG12= E12LG13= E01_BLG14= E05_B

LG

Linkage map of the RILs [MM738 x AG91-25]population

Some regions very rich in distorted markers mean probable chromosome segments from S.aethiopicumancestor ( in AG91-25)

Sequences from GBS were aligned onto the tomato genomic sequence Orthology can be used to identify candidate genes thank to physical map of tomato.

Detection of major gene ERs1 on chromosome 9

ref_26947

novo_30512

novo_77150

novo_32243

novo_4278

novo_41086

novo_61997

novo_5749

novo_1505

emd03C01

CSO475b

COX190a

ref_17276

ref_67868

novo_89055

novo_51578

CMI065a

novo_7810

novo_58873

ref_34168

novo_72335

novo_59329

novo_5688

ref_8676

ref_54787

COX067a

novo_95361

novo_80111

ref_8690

novo_71272

CRO432b

EM134

emf01K21

0

5

10

15

20

9

ERs1

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

ref_26947

novo_30512

novo_77150

novo_32243

novo_4278

novo_41086

novo_61997

novo_5749

novo_1505

emd03C01

CSO475b

COX190a

ref_17276

ref_67868

novo_89055

novo_51578

CMI065a

novo_7810

novo_58873

ref_34168

novo_72335

novo_59329

novo_5688

ref_8676

ref_54787

COX067a

novo_95361

novo_80111

ref_8690

novo_71272

CRO432b

EM134

emf01K21

0

5

10

15

20

9

ERs1

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

Strain LG QTL (cM)length of

CIeggplant

chr.synteny

blockTomato

chr.CMR134 9 106.36 0.360 9 sb35 9GMI1000 9 106.5 0.114 9 sb35 9PSS366 9 106.5 0.114 9 sb35 93strains 9 106.5 0.567 9 sb35 9

Graphic of LOD score according to marker position on LG9

Results of QTL analysis with strains CMR34, GMI1000 and PSS366

ERs1 positioned on the long arm of chromosome 9: Maximum estimated interval= 0,57 cM

Anchor markers with known position on tomato and eggplant reference genomes

Physical position estimated thanks to anchor markers: interval of 1.7 Mbp

ERs1 only detected with strains of phylotype I

ERs1 Phylotype I - specific?

QTL analysis results with strains CFBP3059, CFBP2957, PSS4, TO10

E02

QTL on chromosome 2 detected with 4 strains: PSS4 and TO10 (I), CFBP3059 (III) and CFBP2957 (IIA) Broad-spectrum QTL explaining 12 to 38 % of phenotypic variance QTL on chromosome 5 detected with 2 strains: CFBP2957 and CFBP3059 Major QTL explaining 17 to 50 % of phenotypic variance

E05_b novo_430290,0CDX125a1,9ref_590745,0novo_152146,2novo_324517,8CRX184b8,4CMC184b8,7novo_95239,3novo_700139,6novo_147561_110,2novo_122979_110,8novo_9828913,2novo_49324_114,3CAU275b16,2CAU273a17,4novo_5079127,9novo_6401630,9novo_2339131,9novo_2425632,7novo_4258433,8novo_6931534,6novo_1728437,7

CFB

P2957

CFB

P3059

0

5

10

15

20

25

30

E05_B

novo_717770,0novo_908352,5novo_269683,3novo_703916,1novo_183738,0ref_3373721,7novo_2392747,7novo_9262750,4novo_10162153,3ecm00969,8COF324b70,6COI393a72,7novo_5333574,5CAU393a75,3D_emh02E0876,3novo_480679,7CRC098b80,6novo_7115681,8novo_3016984,7novo_273286,6novo_6679388,8novo_2911096,2ref_50397,6novo_8402103,3novo_86713106,5ref_2107108,6novo_92566109,4novo_76218112,4novo_88131113,3novo_43591114,7novo_95332115,4novo_71535116,7novo_22241120,8novo_33634121,5novo_88421124,3novo_82960125,1novo_35611128,7novo_145307138,7novo_83500143,3novo_25595147,7

CFB

P2957

CFB

P3059

PSS4

TO10

0

5

10

15

20

25

E02

novo_430290,0CDX125a1,9ref_590745,0novo_152146,2novo_324517,8CRX184b8,4CMC184b8,7novo_95239,3novo_700139,6novo_147561_110,2novo_122979_110,8novo_9828913,2novo_49324_114,3CAU275b16,2CAU273a17,4novo_5079127,9novo_6401630,9novo_2339131,9novo_2425632,7novo_4258433,8novo_6931534,6novo_1728437,7

CFB

P2957

CFB

P3059

0

5

10

15

20

25

30

E05_B

novo_717770,0novo_908352,5novo_269683,3novo_703916,1novo_183738,0ref_3373721,7novo_2392747,7novo_9262750,4novo_10162153,3ecm00969,8COF324b70,6COI393a72,7novo_5333574,5CAU393a75,3D_emh02E0876,3novo_480679,7CRC098b80,6novo_7115681,8novo_3016984,7novo_273286,6novo_6679388,8novo_2911096,2ref_50397,6novo_8402103,3novo_86713106,5ref_2107108,6novo_92566109,4novo_76218112,4novo_88131113,3novo_43591114,7novo_95332115,4novo_71535116,7novo_22241120,8novo_33634121,5novo_88421124,3novo_82960125,1novo_35611128,7novo_145307138,7novo_83500143,3novo_25595147,7

CFB

P2957

CFB

P3059

PSS4

TO10

0

5

10

15

20

25

E02

Graphics of LOD score

Straineggplant

chr.QTL position

(cM)length of CI

(cM)synteny

blockTomato

chr.

CFBP30592 71 5 sb06 25 8 3 sb20 12

CFBP29572 78 4 sb06 2

5 5 5 sb20 12

PSS4 2 70.6 4 sb06 2

TO10 2 71 4 sb06 2

Candidate genes for resistance to R. solanacearum

The example of major gene ERs1 on eggplant chr. 9 colocalizing with a cluster of R genes on tomato and potato

Comparison of the synteny block 35 between eggplant transcripts on chr 9 (EO9), tomato genes on chr 9 (T09) and potato genes on chr 9 (P09) (Salgon et al, submitted)

ERs1 interval on chr 9/T09 : 1 CC-NBS-LRR gene 2 RLK gene

ERs1

QTL interval on chr 5/T012 : 4 RLK genes 1 CC-NBS-LRR gene 5 genes coding for

resistance fragment protein

QTL interval on chr 2/T02 : Several clusters of R genes

Heterogeneous Inbred Family (HIF) Material for Near Isogenic Lines (NILs) production and fine mapping of ERs1

Finding SNPs closely linked to the gene ERs1 and defining a narrower interval

To target more precisely candidate genes in the corresponding zone

To use markers more closely linked for breeding

Ongoing: fine mapping of major ERs1 gene

ERs1

1,7 Mb

HIF-1HIF-2HIF-3HIF-4HIF-5

R RRRS

SRRS

R=resistantS=susceptible

b/b homozygous AG91-25a/a homozygous MM738a/b heterozygous

HIF-6HIF-7HIF-8HIF-9

QTL Chr.a Donor parent

Linked marker

Forward primer Reverse primerMarker type

Digestion Enzyme

EBWR2 E02 E6 ecm009ATCTAGTACCATCAAGTCTAAGCAGCA

GTTTAACAACAGCTGAGGCCATGAAA

SSR no -

EBWR9 E09 E6 s_903CCCATTTCACACACAAGCAA

CTCTATTGCCACCCCAAGTG

CAP yes NcoI

Transfer of resistance by SAM in susceptible cultivars BR and BS in Réunion island (strain I-31)

marker CAP s_903 linked to specific major QTL ERs1

marker SSR ecm009 linked to generalist QTL EBWR2

Transfer of resistance by SAM: example of backcross 4

s_903 marker (CAP) used for

transfering ERs1 major gene

ecm009 marker (SSR) used for

transfering EBWR2 QTL

11 individuals carrying both ERs1 and EBWR2 alleles

0

10

20

30

40

50

60

70

80

90

100

0 6 13 20 27 34 41

W (

%)

Days after inoculation

BC4F1(BR*E1)

BC4F1(BR*E2)

BC4F1(BR*E3)

BC4F1(BR*E6)

BC4F1(BS*E1)

BC4F1(BS*E2)

BC4F1(BS*E3)

BC4F1(BS*E6)

E8

E6

Observed % of resistant plants in BC4F1 > theoretical 1:1 ratio

Severe distortion of segregation of marker s_903 in favor of donnor or resistance E6

Selective advantage of genotypes carrying the segments from E6, particularly that one with

major gene ERs1 probably originating from S. aethiopicum

Transfer of resistance by SAM: example of backcross 4

Take Home messages

Resistance to R. solanacearum in eggplant is a complex system with a specific major gene and non-specific QTL,

ERs1 is (at least) phylotype I strains - specific : deployment of resistant breeding lines in areas where phylotype I is predominant,

Transfer of major gene ERs1 in susceptible lines by MAS is effective , but more breeder-friendly markers must be developed from closely linked SNPs sequences,

Cloning ERs1 is a priority for the years to come

ERs1 does not control virulent strains of phylotype I (PSS4, TO10) nor strains of other phylotypes:

need to discover other major genes / QTLs usable by breeders for future creation of broad-spectrum resistance by pyramiding

eggplant

E1. MM853, Dingras multiple purple

E2. MM643, SM6

E3. MM152, Ceylan SM164

E4. MM1752, EG203, Surya

E5. AG91-01, MM931

E6. AG91-25, MM960

E7. MM195 S. linnaeanum (parent of 1rst mapping pop)

E8. MM738 (parent of 1rst mapping population)

E9. S56B, Terong Bulat Hijau

E10. Florida Market, MM 136 (susceptible control)

E1 E3 E4 E9

Mining for new major resistance factors

E8 (susceptible)

Ongoing studies at CIRAD- INRA

X

DH

Aknowledgment

Ralsto team in Réunion Island Main partners

On a local level

On a national level

International level

Financial support

Thank you for your

attention